Rice OsLHT1 Functions in Leaf-to-Panicle Nitrogen Allocation for Grain Yield and Quality

Abstract

Proper allocation of nitrogen (N) from source leaves to grains is essential step for high crop grain yield and N use efficiency. In rice (Oryza sativa) grown in flooding paddy field, amino acids are the major N compounds for N distribution and re-allocation. We have recently identified that Lysine-Histidine-type Transporter 1 (OsLHT1) is the major transporter for root uptake and root-to-shoot allocation of amino acids in rice. In this study, we planted knockout mutant lines of OsLHT1 together wild-type (WT) in paddy field for evaluating OsLHT1 function in N redistribution and grain production. OsLHT1 is expressed in vascular bundles of leaves, rachis, and flowering organs. Oslht1 plants showed lower panicle length and seed setting rate, especially lower grain number per panicle and total grain weight. The concentrations of both total N and free amino acids in the flag leaf were similar at anthesis between Oslht1 lines and WT while significantly higher in the mutants than WT at maturation. The Oslht1 seeds contained higher proteins and most of the essential free amino acids, similar total starch but less amylose with lower paste viscosity than WT seeds. The mutant seeds showed lower germination rate than WT. Knockout of OsLHT1 decreased N uptake efficiency and physiological utilization efficiency (kg-grains/kg-N) by about 55% and 72%, respectively. Taken together, we conclude that OsLHT1 plays critical role in the translocation of amino acids from vegetative to reproductive organs for grain yield and quality of nutrition and functionality.

Keywords: OsLHT1; amino acids; grain quality; grain yield; nitrogen allocation; rice; transporter.

Figure 1

Expression levels of OsLHT1 in rice. Rice (cv. Nipponbare) was grown in paddy field (see materials and methods). (A) The individual organ of the plant at the anthesis stage to be sampled for extraction of total RNA. (B) The relative expression of OsLHT1 quantified by RT-qRCR. Rice housekeeping gene OsActin1 was used as an internal control. Values are means ± SD (n=5 biological replicates). (C–E) GUS staining in root-shoot junction (C), leaf sheath-leaf blade junction (D), and vascular bundles (VB) of mature leaf blades (E). (F) Cross-sections of a leaf blade showing a major (mVB) and minor (miVB) vascular bundles. Scale bar: 100 μm. (G) GUS staining in flower and lemma. (H) GUS staining in three-day-old germinated seeds.

Figure 2

Effect of OsLHT1 mutation on rice growth and grain yield components. Both wild-type (WT, cv. Nipponbare) and Oslht1 mutants were grown in paddy field and harvested at the maturity stage for determining yield components (see Materials and Methods). (A) Image of Oslht1 and WT plants in the field. (B) Mean panicle length. (C) Total grain number per panicle. (D) Total grain weight per panicle. (E) Total grain yield per plant. (F) Mean seed setting rate. (G) Mean 1000-seeds weight. Values are means ± SD (n = 7). Asterisks indicate significant differences between each Oslht1 mutant line and WT detected by one-way ANOVA followed by Tukey’s test. **p < 0.01.

Figure 3

Effect of OsLHT1 mutation on content of total N, total, and individual free amino acid in rice leaf at anthesis and maturity. Both wild-type (WT, cv. Nipponbare) and Oslht1 mutants were grown in paddy field until mature. The flag leaf (fully expanded youngest leaf from top) of WT and Oslht1 plants was sampled for the analysis of total N (A, D), total free amino acids (B, E), and individual free amino acid (C, F). FW, fresh weight. Values are means ± SD (n = 7). Asterisks indicate significant differences between each Oslht1 mutant line and WT detected by one-way ANOVA followed by Tukey’s test. *p < 0.05; **p < 0.01; ns, not significant.

Figure 4

Effect of OsLHT1 mutation on total N content and relative distribution of N in different organs at anthesis and maturity. Both wild-type (WT, cv. Nipponbare) and Oslht1 mutants were grown in paddy field and sampled at anthesis and mature time. (A, C) Total N content in panicles, leaves, culms and total shoot. (B, D) Relative amount of N in vegetative tissues (VT) and reproductive tissues (RT). Leaves and culms were considered as vegetative tissues while panicles were considered as reproductive tissues. Values are means ± SD (n = 7). Asterisks indicate significant differences between each Oslht1 mutant line and wild-type (WT, Nipponbare) detected by one-way ANOVA followed by Tukey’s test. *p < 0.05; **p < 0.01.

Figure 5

Effect of OsLHT1 mutation on content of total N, total and individual amino acid, total crude protein, total starch and amylose, and pasting properties of endosperm starch in harvested grains. Both wild-type (WT, cv. Nipponbare) and Oslht1 mutants were grown in paddy field until mature. (A) Total N concentration. (B, C) Total and individual amino acid content. (D) Total crude protein content. (E) Total starch content. (F) Amylose content. Values in a-f are means ± SD (n = 7). Asterisks indicate significant differences between each Oslht1 mutant line and WT detected by one-way ANOVA followed by Tukey’s test. **p < 0.01; ns, not significant. (G) Pasting properties of endosperm starch. The viscosity value at each temperature is the mean of three replicates. The red line indicates the temperature changes during the measurements.

Figure 6

Effect of OsLHT1 mutation on nitrogen uptake, utilization, and total use efficiency. Both wild-type (WT, cv. Nipponbare) and Oslht1 mutants were grown in paddy field until mature. (A) NUpE: nitrogen uptake efficiency = (total accumulated N/total supplied fertilizer N)*100%. (B) NUtE: nitrogen utilization efficiency = total grain yield/total accumulated N. Values are means ± SD (n = 7). Asterisks indicate significant differences between each Oslht1 mutant line and WT detected by one-way ANOVA followed by Tukey’s test. **p < 0.01.

Figure 7

Effect of OsLHT1 mutation on seed germination, root and shoot growth rate. 100-seeds of each homozygote T4 Oslht1 mutant line and wild-type (WT, Nipponbare) were placed in nutrient-free tap-water. (A) Seed germination rate. DAG, day after germination. (B) Images of seedlings on nutrient-free water on 5 DAG and 12 DAG. Scale bar= 1 cm. (C, D) Root and shoot length of each plant grown in the nutrient-free water for 12 days. Asterisks indicate significant differences between each Oslht1 mutant line and WT detected by the Student’s t-test, **p < 0.01.

Figure 8

Overview model illustrating the OsLHT1 functions in maintaining both N uptake efficiency and N utilization efficiency in rice. In wild type (A), rice roots directly acquire ammonium and amino acids (AAs) from soil. Ammonium in root cells is rapidly assimilated into AAs that are delivered to shoot (sink leaves) in xylem at vegetative stage. Our previous study (Guo et al., 2020) has shown that OsLHT1 (for simplicity, it is indicated by LHT1 in the figure) directly contributes root acquisition and root to shoot transport of a broad spectrum of amino acids. At reproductive stage, large portion of N is re-allocated mainly in the form of amino acids from source leaves to panicles occurring in the phloem. OsLHT1 plays critical role in this process and functions in grain yield and nutrition quality. (B) Knockout of OsLHT1 dramatically reduces N supply from source leaves to developing panicles which are accompanied by reduced shoot biomass and grain yield with higher storage proteins and lower amylose. The higher levels of N in roots or leaves might negatively affect root ammonium uptake in oslht1 plants, probably by a feedback regulatory mechanism, which results in lower N uptake efficiency (NUpE). The limited grain yield by loss of OsLHT1 function results in low N utilization efficiency (NUtE). The sizes of the arrows located to the right of the features analyzed indicate the significant changes in Oslht1 mutants compared with wild-type plants (up, increase; down, decrease).